1. Field of the Invention
This invention relates to a method of selecting the most virulent biotypes from wild-type entomopathogenic viruses and the novel strains of viruses isolated thereby.
2. Description of the Prior Art
One of the impediments to the use of viruses against insect pests is that entomopathogenic viruses act relatively slowly, usually requiring several days or more to cause a lethal infection. Larvae continue to feed until shortly before death; therefore, field applications may not provide adequate crop or foliage protection.
For example, the gypsy moth, Lymantria dispar (L.), is an important forest pest in North America, Europe, and Asia and may cause widespread defoliation. The gypsy moth was responsible for defoliating over 5 million acres (ca. 2.0 million ha) in North America during 1980, over 12 million acres (ca. 4.9 million ha) in 1981, and over 8 million acres (ca. 3.2 million ha) during 1982. Under natural conditions, a nucleopolyhedrosis virus (NPV) is an important mortality factor and may cause population collapse [Glaser, J. Agric. Res. 4: 101-128 (1915); Vasiljevic, Trans. I Int. Congr. Insect Pathol. Biol. Control, Praha, pp. 215-216 (1958); Podgwaite, U.S. Dep. Agric. Handb. No. 539, 15 pp. (1979)]. The virus has been used as a control agent and was registered in the United States as Gypchek [Lewis et al., U.S. For. Serv. Res. Pap. NE-441, 9 pp. (1979a); Lewis et al., U.S. For. Serv. Res. Pap. NE-447, 8 pp. (1979b)] .
Although NPV was successfully used to reduce gypsy moth populations [Rollinson et al., J. Invertebr. Pathol. 7: 515-517 (1965); Injac and Vasiljevic, Plant Prot. 29, No. 143-144: 43-56 (1978); Lewis et al., supra (1979b)], the virus is relatively slow acting, and larvae may continue to feed for almost 2 weeks after exposure. Greater foliage protection and population could occur if viral activity could be increased, either by selection of more desirable isolates [Shapiro and Ignoffo, J. Invertebr. Pathol. 16: 107-111 (1970)] or by the addition of adjuvants [Doane and Wallis, J. Insect. Pathol. 6: 423-429 (1964); Yadava, Z. Angew. Entomol. 65: 175-183 (1970); Shapiro and Bell, Ann. Entomol. Soc. Am. 75: 346-349 (1982)].
Differences in biological activity can be detected among geographical "races" of the same NPV species [Ossowski, J. Insect. Pathol. 2: 35-43 (1960); Smirnoff, J. Insect Pathol. 3: 29-46 (1961); Chauthani et al., J. Invertebr. Pathol. 12: 335-338 (1968); Shapiro and Ignoffo, supra], including the gypsy moth NPV [Magnoler, Entomophaga 15: 407-412 (1970); Rollinson and Lewis, Plant Prot. 24, No. 124-125: 163-168 (1973); Vasiljevic and Injac, Plant Prot. 24, No. 124-125: 169-186 (1973)].
Shapiro et al. [J. Econ. Entomol. 77: 153 (1984)] found that NPV isolated from different geographical locations differed as much as 3000-fold in activity. When these isolates were passed serially four times through L. dispar larvae, the activity of some isolates increased significantly during the second passage. In general, the activities became stabilized from the second to the fourth passage and became similar to the activity of the standard.
Previous studies also reported increases in the biological activities of insect viruses during serial passage [Veber, Colloq. Int. Pathol. Insectes, Paris 3: 403-405 (1962); Smirnoff, J. Insect Pathol. 5: 104-110 (1963); Woodward and Chapman, J. Invertebr. Pathol. 11: 296-301 (1968); Shapiro and Ignoffo, supra]. In the latter case, the pathogenicity of a Baculovirus heliothis isolate increased after five serial passages, and remained unchanged during 11 subsequent passages [Shapiro and Ignoffo, supra]. Serial passage is thought to select a more active isolate from a heterogeneous population, resulting in a more stable, homogeneous virus population [Veber, supra; Shapiro and Ignoffo, supra].
Genotypic variants in wild-type isolates of baculovirus populations are easily recognized by the presence of submolar fragments in the electrophoretic patterns of restriction endonuclease (REN) digestion products. There is a variation in bands of restriction fragments among various geographical isolates, showing both intra-strain and inter-strain heterogeneity among Lymantria dispar NPVs. Moreover, even within different plaque purified isolates of L. dispar NPV, at least five groups could be identified based upon the profile of REN digests. Comparison of the five groupings of plaque isolates having common patterns and virulence show that genotypic variants of L. dispar NPV display differential virulence. These studies have shown that there is a diversity of genomic material within a given wild-type virus [Shapiro and Dougherty, Proc. Symp.: Microbial Control of Spruce Budworms and Gypsy Moths, p. 115 (1985)].